Project Summary Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes epidemics of rheumatologic disease. In the last decade, CHIKV has reemerged to cause outbreaks of unprecedented scale in islands of the Indian Ocean, India, South East Asia, and Europe. In 2013, CHIKV reached the Western Hemisphere, where it is responsible for an ongoing outbreak in the Americas that has affected millions. Large populations of the world are at risk for CHIKV outbreaks due to the expansive range of its mosquito vectors. Up to two thirds of individuals infected with CHIKV experience an incapacitating arthralgia that persists for months or years after the initial infection and is linked to persistent infection of musculoskeletal tissue. Vaccines and antiviral agents are not currently available for CHIKV infection, and treatment is limited to managing symptoms with analgesics and anti-inflammatory drugs. Very little is known about the pathogenesis of CHIKV infection, which is a significant impediment to developing novel prevention or treatment. There is a fundamental gap in our understanding of the mechanisms that facilitate CHIKV persistence and chronic disease. Addressing this gap has the potential to guide CHIKV vaccine design and provide new therapeutic targets for the treatment of acute and chronic CHIKV disease. This proposal takes advantage of attenuated and pathogenic CHIKV strains that differ in their ability to establish persistence in musculoskeletal tissue of infected mice. The central hypothesis is that efficient T follicular helper (TFH) cell responses contribute to clearance of the acute CHIKV strain 181/25 and that impairment of TFH cell responses promotes persistence of the pathogenic CHIKV strain AF15561 in an otherwise immunocompetent host. Guided by substantial preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) To define TFH and germinal center (GC) B cell differentiation during acute and chronic CHIKV infection, 2) Determine whether the function of TFH cells is altered during pathogenic CHIKV infection, and 3) Define the requirement for TFH cells in CHIKV clearance. Under the first aim, the magnitude of the TFH, T follicular regulatory (TFR; a specialized follicular T cell population that regulates TFH cell functions), and GC B cell response during acute and chronic CHIKV infection will be measured by flow cytometry analysis of endogenous cells and transferred TCR transgenic T cells. Under the second aim, the quality of the TFH and GC B cell response during acute and chronic CHIKV infection will be measured by response to immunizations, molecular analysis of isolated TFH and TFR cells, and in vitro TFH stimulation assays. In the final aim, mice that lack TFH cells will be infected to determine whether the absence of TFH cells converts an acutely cleared CHIKV strain to a chronic infection. The proposed research is significant because it will provide new insight into the immunologic mechanisms that facilitate persistence of CHIKV in musculoskeletal tissue. Ultimately, this knowledge will provide an important foundation for the development of vaccines and novel therapeutics that may improve the prevention and treatment of CHIKV infection.